Antibodies to adipose tissues

ABSTRACT

A non-therapeutic method of producing antibodies that bind adipose tissues in a target animal in need of the antibodies for modulating the content of adipose tissues in the target animal, the method comprising the steps of (a) preparing an antigen from the adipose tissues of a source animal, (b) administering the antigen to an egg-laying animal to cause production of the antibodies, and (c) obtaining the antibodies from eggs of the egg-laying animal wherein the source animal and the egg-laying animal belong to different species.

FIELD OF INVENTION

The present invention relates to a method of producing antibodies thatbind adipose tissues and in particular polyclonal antibodies that bindto characterizing components of the plasma membrane in adipose tissuesin animals (e.g. farm animals) and/or humans. The present invention alsorelates to antibodies obtainable according to the method, use of theantibodies, a feed additive comprising the antibodies, a medicamentcomprising the antibodies, and a method of modulating content of adiposetissues in the body of a target animal in need of the antibodies.

BACKGROUND OF THE INVENTION

In animal farming, one of the main objectives is to increase the growthrate of the farm animals such that under generally the same conditionsof husbandry, the animals will grow faster and as such productivity ofthe animal farm can be increased. In the past, before modern technologyhas been adopted in animal farming, farmers would normally simply feedthe animals with more food in the hope that higher food consumptionwould cause the animals to grow and increase in weight faster. However,there is a limit that such method can help in increasing the body weightof the animals. Besides, the drawback is that this method would increasethe total consumption of animal feed and accordingly undesirablytranslate to higher operation costs.

Another method to promote growth in farm animals is to administer growthhormones to the animals. This method is however undesirable for a numberof reasons. Firstly, growth hormones from different animals are seldomhomogenous and different mammalian animals, for example, only react tocertain types of specific growth hormones. Since suitable exogenousgrowth hormones are normally extracted from pituitary glands, it israther difficult and uneconomical to prepare sufficient quantity ofsuitable exogenous growth hormones for use on a large-scale application.Although exogenous growth hormones can now be prepared using DNArecombinant technology, exogenous growth hormones manufactured by suchmethod are still rather expensive. Secondly, the administration ofexogenous growth hormones into farm animals is normally performed bydirect injection, which is inevitably rather costly and difficult toadminister in a large farm with animals in tens of thousands. Thirdly,it is rather difficult to control the dose administered to produceprecisely the desired effect, and an overdose of exogenous growthhormones is likely to be harmful to the animals. Fourthly, residuals ofthese exogenous growth hormones may be passed to the meat products andsubsequently to humans through consumption thereof. Further studies inthis regard are required although some scientists are concerned aboutthe negative side effects of these exogenous growth hormones to humans.

Various feed additives have also been proposed to be added to animalfeed such that animals fed with these feed will grow faster.Unfortunately, regardless of which of the above methods is used, it isoften the case that a relatively large percentage of the increased bodyweight results from an increase in fat content and not from lean musclecontent. This problem is particularly prominent in swine although otherfarm animals experience similar problems. As humans have become morehealth conscious nowadays, there is little demand, if any, for meatproducts having a high fat content. There is therefore a growing demandfor meat products having as low a fat content as possible (i.e. highcontent of lean muscle).

Numerous methods have been proposed to cause farm animals to developwith higher muscle content. A very old method is to raise the animals inan open or semi-open farm such that the animals would have moreopportunity to exercise such that the fat content in their body may bereduced. However, this method is nearly impossible to carry out inpractice in modern farms wherein space is at a premium. Besides, thismethod is rather unpredictable. Animals subjected to this method maystill have a rather high fat content in their body.

Hence, there continues to exist a need for a substance for regulatingand reducing the fat content in farm animals. Preferably, the substanceshould be easy to administer and natural, and should not have any sideeffects similar to those caused by artificial or exogenous growthhormones. In other words, the substance should be safe to administer. Asubstance, which works in farm animals, should preferably also work inhumans with modifications.

It is thus an object of the present invention in which the above issuesare addressed, or at least to provide a useful alternative to thepublic.

SUMMARY OF INVENTION

According to a first aspect of the present invention, there is provideda non-therapeutic method of producing antibodies that bind adiposetissues in a target animal in need of the antibodies for modulating thecontent of adipose tissues in the target animal, the method comprisingthe steps of (i) preparing an antigen from the adipose tissues of asource animal, (ii) administering the antigen to an egg-laying animal tocause production of the antibodies, and (iii) obtaining the antibodiesfrom eggs of the egg-laying animal wherein the source animal and theegg-laying animal belong to different species.

The antibodies are to be used to bind characterizing components ofplasma membrane of the adipose tissues of the target animal. Inparticular, the antibodies may bind granular viscosity proteins and/orfiber viscosity proteins of the adipose tissues of the target animal.The antibodies suitably specifically bind to the characterizingcomponents. By specifically binding to the charactering components, itmeans that the antibodies have low, or no noticeable, cross-reactivity.

The method comprises a step of causing production of the antibodies fromwithin the body of the egg-laying animal. The method advantageouslycomprises a step of causing deposition of the antibodies to the eggs ofthe egg-laying animal. In particular, the step of obtaining theantibodies from the eggs may comprise a step of isolating the antibodiesfrom the egg yolk of the eggs.

The antigen suitably comprises plasma membrane, its adipocyte plasmamembrane surface proteins, or fragments thereof, of the adipose tissuesof the source animal.

The antibodies are preferably polyclonal antibodies.

Preferably, the source animal and the egg-laying animal belong todistinctly different species. The egg-laying animal is preferably anavian animal and the source animal may be a non-avian animal. Inparticular, the egg-laying animal may be a hen and the source animal maybe a mammal such as a swine.

Preferably, the target animal and the egg-laying animal belong todifferent species. More preferably, the target animal and the egg-layinganimal belong to distinctly different species. By distinctly differentspecies, it means they are phylogenetically distinct. For example, aswine is a mammal and a hen is an avian, and they are distinctlydifferent species.

Preferably, the target animal and the source animal belong to a samespecies. Alternatively, the target animal and the source animal belongto closely related species. By closely related species, it means thatthey are phylogenetically related. For example, a swine and a rat areclosely related species and they are both mammals and the antibodiesproduced in response to the antigen prepared from the adipose tissues ofa swine would work sufficiently well in modulating the content of theadipose tissues in a rat.

In one embodiment, the target animal is a farm animal. In particular,the source animal and/or said target may a swine. The fat content of aswine is often higher than other farm animals and there may be asignificant reduction of adipose tissues in the swine administered withthe antibodies. However, the target animal may be any other animal whichrequires modulation of its adipose tissues. For example, the targetanimal may be a cow for producing leaner beef.

After the method one or all of the animals may be sacrificed.

In another embodiment, the target animal is a patient.

According to a second aspect of the present invention, there areprovided antibodies obtainable according to the method defined above.According to a third aspect of the present invention, there are providedthe antibodies for use in a method of treatment or diagnosis.

According to a fourth aspect of the present invention, there is providedthe use of antibodies defined above for the manufacture of a medicamentfor the treatment of a condition caused by an excess of adipocytes.

According to a fifth aspect of the present invention, there is provideda feed additive comprising an effective amount of antibodies definedabove. In particular, the feed additive may be adapted to lower thecontent of the adipose tissues in the target animal.

In one embodiment, the feed additive may comprise egg yolk of the eggscontaining the antibodies.

According to a sixth aspect of the present invention, there is provideda medicament comprising a pharmaceutically effective amount ofantibodies defined above. Preferably, the medicament is adapted to beadministered via ingestion. Alternatively, the medicament is adapted tobe administered via injection.

According to a seventh aspect of the present invention, there isprovided a method of modulating content of adipose tissue in the body ofa target animal in need of antibodies, comprising the steps of (i)preparing an antigen from adipose tissues of a source animal, (ii)administering the antigen to an egg-laying animal, (iii) allowing theantibodies to be produced by the egg-laying animal in response to theantigen, (iv) obtaining the antibodies from eggs of the egg-layinganimal, and (v) administering a pharmaceutically effective amount of theantibodies to the target animal, wherein the source animal and theegg-laying animal belong to different species. In particular, the stepof obtaining may comprise a step of isolating the antibodies from theegg yolk of the eggs.

The method preferably comprises a step of administering the antibodiesvia ingestion. Alternatively, the method comprises a step ofadministering the antibodies via injection.

The modulation method comprises a step of binding the antibodies tocharacterizing components of plasma membrane of the adipose tissues inthe target animal. The method may comprise a step of binding theantibodies to granular viscosity proteins of the adipose tissues in thetarget animal. The method may also comprise a step of binding theantibodies to fiber viscosity proteins of the adipose tissues in thetarget animal. In particular, the antibodies suitably specifically bindto the characterizing components. By specifically binding to thecharactering components, it means that the antibodies have low, or nonoticeable, cross-reactivity.

Preferably, the antigen comprises plasma membrane, its adipocyte plasmamembrane surface proteins, or fragments thereof, of the adipose tissuesof the source animal.

The antibodies are preferably polyclonal antibodies.

Preferably, the source animal and the egg-laying animal belong todistinctly different species. The egg-laying animal is preferably anavian animal and the source animal may be a non-avian animal. Inparticular, the egg-laying animal may be a hen and the source animal maybe a mammal such as a swine.

Preferably, the target animal and the egg-laying animal belong todifferent species. More preferably, the target animal and the egg-layinganimal belong to distinctly different species. By distinctly differentspecies, it means they are phylogenetically distinct. For example, aswine is a mammal and a hen is an avian, and they are distinctlydifferent species.

Preferably, the target animal and the source animal belong to a samespecies. Alternatively, the target animal and the source animal belongto a closely related species. By closely related species, it means thatthey are phylogenetically related. For example, a swine and a rat areclosely related species and they are both mammals and the antibodiesproduced in response to the antigen prepared from the adipose tissues ofa swine would work sufficiently well in modulating the content of theadipose tissues in a rat.

The modulation method may be a non-therapeutic method.

In one embodiment, the target animal is a farm animal. In particular,the source animal and/or the target may be a swine.

The egg-laying animal is preferably an avian animal and the sourceanimal may be a non-avian animal. In particular, the egg-laying animalmay be a hen and the source animal may be a mammal such as a swine.

After the method one or more of the animals may be sacrificed.

In another embodiment, the target animal is a patient.

According to an eighth aspect of the present invention, there isprovided a method of producing antibodies that bind adipose tissues in atarget animal in need of the antibodies for modulating the content ofadipose tissues in the target animal, the method comprising the steps of(i) preparing an antigen from the adipose tissues of a source animal,(ii) administering the antigen to an egg-laying animal to causeproduction of the antibodies, and (iii) obtaining the antibodies fromeggs of the egg-laying animal wherein the source animal and theegg-laying animal belong to different species.

According to a further aspect of the invention, there is providedantibodies that specifically bind adipose tissues in a target subjectwhich is a farm animal or a patient in need of the antibodies formodulating the content of the adipose tissues in the target subject. Theantibodies bind to characterizing components of plasma membrane of theadipose tissues. In particular, the antibodies may bind to granularviscosity proteins of the adipose tissues. The antibodies may bind tofiber viscosity proteins of the adipose tissues.

The antibodies are obtained from and/or comprised in eggs of anegg-laying animal. The antibodies are deposited to the eggs of theegg-laying animal. The antibodies are produced from within the body ofthe egg-laying animal. The antibodies are produced in response to anantigen administered to the egg-laying animal.

The antigen is prepared from adipose tissues of a source animal. Theantigen comprises plasma membrane and/or its adipocyte plasma membranesurface proteins of the adipose tissues of the source animal.

Preferably, the target subject and the source animal belong to a samespecies. Alternatively, the target subject and the source animal belongto closely related species.

Preferably, the source animal and the egg-laying animal belong todistinctly different species.

Preferably, the antibodies are polyclonal antibodies.

According to a further aspect of the present invention there is provideda feed additive comprising an effective amount of the antibodies definedabove. The feed additive is adapted to lower the content of the adiposetissues in the target subject.

According to a further aspect of the present invention, there isprovided a medicament comprising a pharmaceutically effective amount ofthe antibodies defined above. The medicament may be adapted to beadministered via ingestion. The medicament may be adapted to beadministered via injection.

According to a further aspect of the present invention, there isprovided a method of modulating content of adipose tissues in the bodyof a target subject which is a farm animal or a patient in need ofantibodies comprising a step of administering a pharmaceuticallyeffective amount of the antibodies that specifically bind the adiposetissues in the target subject.

The method comprises a step of binding the antibodies to characterizingcomponents of plasma membrane of the adipose tissues. In particular, themethod may comprise a step of binding the antibodies to granularviscosity proteins of the adipose tissues. The method may comprise astep of binding the antibodies to fiber viscosity proteins of theadipose tissues.

The method comprises a step of administering of said composition viaingestion.

The antibodies are polyclonal antibodies.

According to a further aspect of the present invention, there isprovided a method of manufacture a composition comprising apharmaceutically effective amount of the antibodies defined above,comprising a step of obtaining the antibodies from eggs of an egg-layinganimal. The method preferably comprises a step of allowing deposition ofthe antibodies to the eggs of the egg-laying animal.

The method preferably comprises a step of causing production of theantibodies from within the body of the egg-laying animal. In particular,the method comprises a step of causing production of the antibodies inthe egg-laying animal in response to an antigen prepared from adiposetissues of a source animal. The method comprises a step of administeringthe antigen to the egg-laying animal.

Preferably, the antigen comprises plasma membrane and/or its adipocyteplasma membrane surface proteins of the adipose tissues of the sourceanimal.

Preferably, the target subject and the source animal belong to a samespecies. Alternatively, the target subject and the source animal belongto closely related species.

Preferably, the source animal and the egg-laying animal belong todistinctly different species.

Preferably, the antibodies are polyclonal antibodies.

The composition may comprise egg yolk containing said antibodies.

DETAILED DESCRIPTION OF THE INVENTION

A biological substance (e.g. growth hormone) may be produced andextracted from the pituitary glands of a “production” animal. Byproduction animal it means the animal is used as a machinery to producethe desired biological substance. Depending on the type or nature of thebiological substance, they may actually be obtained or isolated usingdifferent methods. For instance, if the biological substance is a growthhormone which is present in the colostrum of cow milk in a productionanimal, an appropriate isolation procedure of the growth hormonetherefrom is to be performed. Alternatively, if the growth hormone ispresent in the blood serum in a production animal, an alternativesuitable isolation procedure of the growth hormone therefrom is to beperformed. However, whichever artificial isolation procedure is used, ithas been found that isolation of a sufficient quantity of biologicalsubstance of interest for commercial use from an animal source is verydifficult. The difficulty arises firstly because the quantity ofbiological substance produced is usually very small. Secondly, isolationof biological substance from the animal is very costly. The samedifficulty similarly exists in the extraction or isolation of specificantibodies of interest from a production animal.

In the present invention, it is demonstrated that antibodies prepared inaccordance with the present invention when administered to a targetanimal, which may be a farm animal, reduce or at least modulate theoverall fat content in its body to a more desired level and thus produceleaner meat. When the present invention is applied for use in humans,the target animal means a patient in need of the antibodies.

Generally, adipose tissues are firstly removed from a source animal.Plasma membrane of the adipose tissues is then isolated from the adiposetissues. The isolated plasma membrane includes all its adipocyte plasmamembrane proteins and recognition sites such as granular and fiberviscosity proteins. The isolated plasma membrane is used to prepare asubstance for use as an antigen. The substance is preferably in a formsuitable for injection and is engineered to have an immunologicallyeffective concentration of the antigen which is adapted to elicit adesired immunological response in a production animal.

In the present invention, the substance is administered to theproduction animal which is an egg-laying animal such as a hen. The useof hen as a production animal is particularly preferable because a hennormally produces more eggs than other egg-laying fowl. For instance, anaverage hen in a commercial farm can often lay as many as 200 to 300eggs per year. The amount of egg yolk produced by a hen is accordinglyvery significant. However, other egg-laying animals such as ducks mayalso be used.

Once the substance containing the antigen is administrated to theproduction animal such as by injection, the body of the productionanimal will react and initiate an immune response to the antigen byproducing antibodies. As described above, the antigen of theadministered substance actually comprises the plasma membrane of theadipose tissues, or fragments thereof, from the source animal. Theantibodies produced by the egg-laying animal are thus polyclonal andadapted to bind various different characterizing components, i.e. theadipocyte plasma membrane proteins of the plasma membrane. During theresearch and development of the present invention, it has beenidentified that a relatively significant amount of the antibodiesproduced within the body of the production animal are deposited in theeggs which are subsequently laid by the production animal. It hasfurther been identified that the egg yolk of the eggs has a much higherconcentration of the antibodies than the egg white indicating that thereis a preferential deposition of the antibodies in the egg yolk. In otherwords, the problem of producing and isolating a biologically usefulsubstance from an animal source is addressed in the context of thepresent invention. In particular, the eggs can be seen as a warehousefrom which the antibodies of interest can be retrieved relativelyeasily. It is also for this reason that a hen is preferably used as aproduction animal because of the relatively large number of eggs thatcan be produced therefrom.

The produced antibodies can then be isolated from the egg yolk.Alternatively, the raw egg yolk containing the antibodies may be useddirectly or after processing such as by subjecting it to desiccation toform egg yolk powder. An effective amount of the isolated antibodies,the raw or processed egg yolk containing the antibodies is thenadministered to a target animal. One main application of the presentinvention is intended to be in animal farming and in this case thetarget animal may be a farm animal. However, as indicated above, thepresent invention may also be applied for use in humans and thus thetarget animal may be a patient in need of the antibodies.

When administered to the target animal, the antibodies will bind tocharacterizing structures or domains (e.g. the surface proteins of cellsin the target animal) which are similar to the adipocyte plasma membraneproteins of the adipose tissues of the source animal. For instance, ifthe source animal is a swine and the target animal belong to the samespecies of swine, the administered antibodies will bind to the adipocyteplasma membrane proteins of the adipose tissues in the body of thetarget animal and interfere with the physiological development of itsadipose tissues. It has been identified during the research anddevelopment of the present invention that such binding and/orinterference significantly decrease the content of adipose tissues inthe target animal both in terms of its weight percentage and absoluteweight.

As indicated above, the source animal and the target animal may belongto the same species of animals. The more closely related of the sourceanimal and the target animal are, the more effective the producedantibodies for targeting adipose tissues of the target animal andeventually reducing or at least modulating the fat content in the bodyof the target animal. However, the source animal and the target animalneed not belong to identical species. For example, the source animal maybe a cow but the target animal may be a swine. Since both cows and swineare mammals, their adipose tissues and in particular the plasma membranethereof have more resemblance than between for example the adiposetissues of an avian and a mammal. In summary, the more closely relatedthe source animal and target animal are, the more effective the producedantibodies are in binding, interfering, modulating and/or reducing thefat content in the body of the target animal.

It is however to be noted that the source and production animals shouldpreferably be sufficiently different. Otherwise, the substancecontaining the antigen administered to the production animal would notelicit an effective immunological response to produce a sufficientamount of antibodies of interest. For instance, if the source animal isa duck, the antigen prepared from its adipose tissues will elicit arelatively low immunological response in a hen since both the duck andthe hen are avian animals and their adipose tissues are relativelysimilar. The source animal and the production animal should be differentbecause an animal will not readily produce antibodies to antigen that itconsiders to be “self”.

The present invention is described in further detail by way of thefollowing experiments.

EXPERIMENTS

Experiment I: Procedures for Producing Antibodies to Adipose Tissues ofSwine in Hens

A. Isolation of Plasma Membrane from Adipose Tissues of a Source Animal

Adipose tissues were removed from the back of a source animal. Thesource animal used in the experiment was an Erhualian pig. The adiposetissues were treated and homogenized in an extraction medium at around37° C. in a Waring blender at 2000 rpm for 5 min and then treated withultrasound for 10 minutes. The extraction medium was made of 0.25M ofsucrose, 0.01M of Na₂HP0₄, 0.002M EDTA, 0.2 mM PMSF and adjusted topH7.4 at 40° C. The homogenate was then centrifuged at 5000 rpm for 30minutes at 37° C. to separate the triglyceride from the othercomponents.

The supernatant containing the triglyceride was then removed aftercentrifugation and the remainder, i.e. the infranatant, was subjected tocentrifugation at 10000 rpm for 30 min at 4° C. The supernatant thereofwas then subjected to centrifugation at 10000 rpm for 30 minutes at 4°C. and the supernatant was retained. The supernatant was then subjectedto centrifugation at 38000 rpm for 1 hour at 4° C. The plasma membraneincluding its adipocyte membrane proteins from the adipose tissues wasobtained. The membrane proteins were then stored at −20° C. until theywere used.

B. Production of Antibodies to Pig Adipocyte Plasma Membrane and itsProteins

The plasma membrane obtained from the above procedure was used toprepare an antigen to elicit immune response from a production animal.In this experiment, egg-laying hens were used as the production animal.In the experiment, an initial injection comprising the antigen wasprepared to contain approximately 80 μg of the plasma membrane and itsproteins initially suspended in 0.5 ml of complete Freund's adjuvant. Asecond injection comprising the same antigen suspended in incompleteFreund's adjuvant for boosting the immune response was subsequentlyadministered also by direct injection. Each round of administration wasperformed in at least 20 different intercutaneomucous sites of the hensat intervals of once every four weeks. After the third and fourthbooster injections, egg yolk was subsequently obtained from eggs laid bythe hens. Antibody responses of the egg yolk were then assessed.

C. Enzyme Immunoassay of Egg Yolk Antibodies to Plasma Membrane of PigAdipocytes

The egg yolk antibodies were prepared and screened for antibody titeragainst a suitable adipocyte plasma membrane, and for cross reactivitywith liver, kidney, red blood cells and skeletal muscle by ELISA. 100 μlof the plasma membrane containing 0.25 μg of the adipocyte plasmamembrane proteins in carbonate-buffered solution was coated onto eachwell of 96-well polystyrene plates. The plates were kept overnight at 4°C. in a humidified chamber. The wells were then emptied and blocked withPBS containing 0.05% Tween 20 for three times. 100 μl of the egg yolkdiluted in PBST was added to each well. The plates were kept for 1 hourat 37° C. and subjected to washing with PBST for three times. 100 μl ofrabbit anti-chicken 1 gG HRP conjugate diluted to 1:5000 in PBST wasadded to each well. The plates were incubated for 1 hour at 37° C. Theplates were washed three times with PBST. 100 μl of O-phenylenediamin(OPD) substrate (1.5 mg/ml) was then added to each well. The plates thenwere incubated at 37° C. for 5 to 10 minutes, and the reaction in eachwell was stopped with 50 ul of 2M H₂S0₄. Absorbance was measured at 490nm using an ELA plate reader. Each assay was performed in duplicate andrepeated three times. It was found that the titer of the antibodies inthe egg yolk was more than 1:12800 which is considered as a relativelyhigh titer value in the context of the present invention.

Experiment II: Effect of Adipose Tissues Antibodies on Body Weight ofTarget Animal

A. Background

The target animal used in this experiment was laboratory rats.Ninety-six female growing rats were used in the experiment with anaverage body weight of 140 g. The rats were divided equally and randomlyinto four groups. The rats were kept in sub-groups of three in cages.The rats were fed with regular rat feed. The experiment commenced on 30Sep. 2001 and ended on 14 Dec. 2001.

B. Procedure

The four groups of rats consist of two test groups and two respectivecontrol groups. The two test groups include a first test group in whicheach rat was subjected to injection of raw egg yolk containing theadipose tissue antibodies subcutaneously in different locations at theirback. The antibodies are produced in accordance with the procedures ofExperiment I above. In particular, the antibodies are produced inresponse to the injection of an antigen prepared from the adiposetissues of a pig. The egg yolk adipose tissue antibodies were obtainedbased on similar procedures described in the above Experiment I. Thedose of each injection was 1 ml per rat per day. Each round ofadministration includes one injection each day for four consecutivedays. The egg yolk was administered again once a month during theexperiment. The titer of the antibodies in the raw egg yolk was morethan 1:12800.

The second test group was administered with the same dose, concentrationand frequency of the egg yolk adipose tissue antibodies but by oralingestion instead of injection.

There is a corresponding control group for each of the two test groupsof rats. The control groups of rats were administered with regular rawegg yolk.

C. Results

The following tables show the results of the experiment. TABLE 1 Effectsof the egg yolk adipose tissue antibodies on body weight and feedconversion rate. (X ± SE) Beginning Ending Body Feed body body weightFood conversion weight (g) weight (g) gain (g) intake (g) rate Firsttest 163.42 ± 297.64 ± 133.91 ± 22.25 ± 6.25 ± group (by 2.55 5.23 4.230.23 0.20 injection) First 162.88 ± 289.00 ± 126.62 ± 21.87 ± 6.27 ±control 2.28 5.33 4.40 0.26 0.30 group (by injection) Second 159.10 ±281.56 ± 122.25 ± 21.75 ± 5.87 ± test group 2.70 7.43 5.02 0.23 0.22 (byingestion) Second 164.21 ± 292.82 ± 127.18 ± 21.72 ± 6.52 ± control 2.006.54 6.20 0.52 0.21 group (by ingestion)

TABLE 2 Effects of the egg yolk adipose tissue antibodies on fat contentin various parts of the rat body (X ± SE) Omental and mesentericParamentrial Perirenal Gastrocnemius fat Content fat Content fat Contentfat Content (%) (%) (%) (%) First test group 18.06 ± 0.72^(Aa) 26.43 ±1.72^(Aa) 17.95 ± 1.48^(Aa)   6.03 ± 0.11^(a) (by injection) Firstcontrol group 18.75 ± 0.87^(Aa) 27.58 ± 1.78^(Aa) 19.18 ± 1.32^(Aa) 5.73 ± 0.06^(b) (by injection) Second test group 14.22 ± 1.02^(Bb)18.63 ± 1.98^(Bb) 12.01 ± 1.17^(Bb) 5.89 ± 0.11 (by ingestion) Secondcontrol group 17.16 ± 1.05^(a)   24.58 ± 2.24^(a)   15.32 ± 1.25^(a)  5.83 ± 0.09 (by ingestion)KEY:Values bearing different superscripts are significantly different;^(A,B)means P < 0.01;^(a,b)means P < 0.05

TABLE 3 Effects of the egg yolk adipose tissue antibodies on level oftriglyceride, cholesterol and fatty acids in blood of the rat body (X ±SE) Total Triglyceride cholesterol Total fatty (mg/dl) (mg/dl) acids(μmol/L) First test 33.83 ± 1.70^(Aa) 61.05 ± 3.56 140.69 ± 9.73 group(by injection) First control 45.42 ± 2.67^(B)   58.91 ± 2.44 135.29 ±7.31 group (by injection) Second test 32.00 ± 1.60^(Aa) 61.35 ± 2.61  161.21 ± 8.05^(A) group (by ingestion) Second control 41.20 ±2.48^(b)  54.64 ± 4.21  121.72 ± 7.47^(B) group (by ingestion)KEY:Values bearing different superscripts are significantly different;^(A,B)means P < 0.01;^(a,b)means P < 0.05D. Conclusion and Discussion

In Table 1, it is shown that the administration of the antibodies byinjection increased the weight gain and the food consumption in thefirst test group of rats when compared to the corresponding controlgroup by 5.8% and by 1.7% respectively. The feed conversion rate washowever decreased by 0.32%. It is also shown that the administration ofthe antibodies by ingestion when compared to the corresponding controlgroup decreased the weight gain in the second test group of rats by 3.9%and increased the food consumption by 0.14%. The feed conversion ratewas decreased by about 10%. The experimental data in relation to thefirst test and control groups illustrates that the administration of theantibodies by injection increased the body weight slightly although thefeed conversion rate was lowered very slightly. A low feed conversionrate means that less amount of feed is required to produce a unit ofbody weight. The experimental data in relation to the second and testand control groups illustrates that the administration of the antibodiesby ingestion decreased the body weight gain slightly and the feedconversion efficiency was substantially decreased by 10%. This isimportant and demonstrates that the administration of the antibodiesthrough ingestion is more effective in reducing the overall body weightslightly and lowering the feed conversion rate very significantly.

Referring to Table 2, it is shown that the administration of theantibodies by injection caused to the fat content of their omental andmesenteric, paramentrial and perirenal tissues to decrease by 3.7%, 4.2%and 6.4% respectively when compared to the corresponding control groups.However, the fat content of the gastrocnemius was increased by 5.2%. Itis also shown that the administration of the antibodies by ingestionvery significantly decreased the fat content of their omental andmesenteric, paramentrial and perirenal tissues by 17.1%, 24.2% and 2.16%respectively when compared to the corresponding control group.

As clearly shown in this experiment, the administration of theantibodies by whichever means, injection or ingestion, is generallyeffective in reducing the fat content in various parts of the body inthe animal. In particular, it is shown that administration way ofingestion is significantly more effective in reducing the general fatcontent of the animal.

Referring to Table 3, it is shown that the administration of theantibodies by injection caused the level of triglyceride to decreasesignificantly by 25.5%. The levels of cholesterol and free fatty acidswere caused to rise marginally by 3.6% and 4.0% respectively. Inrelation to the administration of the antibodies through oral ingestion,the level of triglyceride was caused to decrease also significantly by22.3%. The levels of cholesterol and free fatty acids were caused toincrease by 12.3% or 32.4 respectively.

When the data of all three tables are considered together, it is clearlyshown that the administration of the antibodies into the animal doesreduce the overall fat content in its body and this is supported by thedecrease in the overall fat content in the test groups of rats shown inTable 2 and the levels of triglyceride shown in Table 3. In particular,it is shown that administration of the antibodies by means of oralingestion is more effective when compared to that by direct injection.

The above results are significant in two ways. Firstly, surprisingly,the antibodies produced according to the present invention are moreeffective when administered orally. This is important because theantibodies can in principle be mixed with a standard feed material inanimal farming and as such administration thereof will become very easy,effective and yet can achieve its intended function in reducing fatcontent. Secondly, there are no observable side effects to the animal.For instance, the overall body weight is not affected in any significantway and yet the fat content is reduced. The feed conversion rate is alsoslightly improved. In other words, there is less fat content and higherlean meat content in the body of the target animal.

In table 3, it is shown that the level of free fatty acids was increasedsignificantly. This can be explained as follows. Triglyceride iscomposed of fatty acids and glycerol. When the level of triglyceride(i.e. fat content) is caused to be reduced, the equilibrium is shiftedto the right as illustrated below.Triglyceride  <=>  fatty  acids + glycerol→

For this reason, the level of free fatty acids was caused to increase.

Experiment III: Effect of Adipose Tissues Antibodies on Body Weight ofRats

A. Background and Procedure

The target animal used in this experiment was laboratory rat. Onehundred fifty female growing rats were used in the experiment with anaverage body weight of 110 g. The rats were randomly divided into fivegroups (i.e. Groups I to V) in cages with each cage keeping three rats.Group I was the control group and Groups II to V were the test groups.The experiment was preceded by one week feeding the rats with a regularfeed. The composition of the regular feed is as follows. TABLE 4Composition of the regular feed Ingredients Wt % Proteins 24.02 Fats3.94 7.9 Calcium 1.4 Phosphorus 0.8 Salts 1.31

During the experiment, the Group I rats were fed with the regular diet.The Groups II to V rats were fed with the regular diet added withdifferent quantities of adipose tissues antibodies prepared inaccordance with the method described in Experiment I above. Theantibodies are produced in accordance with the procedures of ExperimentI above. In particular, the antibodies are produced in response to theinjection of an antigen prepared from the adipose tissues of a pig.

The experiment began on 28 Nov. 2002 and ended on 18 Feb. 2003.

B. Results TABLE 5 Effect of antibodies on adipose tissues (x ± sd)Weight\ Antibodies in diet Control 75 ppm 500 ppm 1000 ppm 6000 ppmBeginning 115.19 ± 115.24 ± 114.72 ± 114.81 ± 115.65 ± body weight 6.787.81 7.54 6.65 9.19 (g) Final body 304.13 ± 302.23 ± 296.77 ± 296.66 ±304.00 ± weight 14.65 21.38 22.68 23.17 23.16 Weight gain 188.94 ±187.00 ± 182.04 ± 181.68 ± 188.05 ± (g) 16.37 21.75 21.52 25.09 18.92 Pvalue 0.70 0.17 0.19 0.84 Food intake 24.68 ± 24.42 ± 23.93 ± 25.53 ±25.90 ± (g/day/rat) 2.18 2.63 1.90 2.17 1.33 P value 0.81 0.42 0.39 0.15Parametrial 6.04 ± 5.02 ± 5.33 ± 5.97 ± 5.14 ± fat (g) 1.66 1.72 1.932.10 1.79 P value 0.025 0.137 0.887 0.053 Parametrial 19.80 ± 16.44 ±18.11 ± 20.00 ± 16.90 ± fat index 5.20 4.96 6.27 6.69 5.24 P value 0.0140.266 0.898 0.041 Mesenteric 4.64 ± 4.28 ± 4.42 ± 4.44 ± 4.15 ± fat (g)0.86 0.99 0.96 1.16 1.00 P value 0.149 0.362 0.458 0.052 Mesenteric15.21 ± 14.20 ± 14.98 ± 14.82 ± 13.69 ± fat index 2.56 2.80 2.89 2.982.74 P value 0.155 0.739 0.588 0.033 Perirenal 4.80 ± 4.23 ± 4.06 ± 4.01± 3.83 ± fat (g) 1.39 1.47 1.37 1.35 1.37 P value 0.144 0.054 0.0410.013 Perirenal 15.79 ± 13.90 ± 13.85 ± 13.55 ± 12.66 ± fat index 4.404.36 4.54 4.46 4.35 P value 0.113 0.115 0.071 0.011 Celiac fat 15.48 ±13.53 ± 13.74 ± 14.21 ± 13.46 ± (g) 3.36 3.79 3.80 4.67 4.07 P value0.041 0.069 0.240 0.044 Celiac fat 50.76 ± 44.53 ± 46.77 ± 47.72 ±44.331 index 10.25 10.67 12.20 14.21 11.94 P value 0.026 0.182 0.3520.032 Gastroc- 1.85 ± 1.85 ± 1.81 ± 1.83± 1.87 ± nemius 0.11 0.13 0.120.17 (g) P value 0.946 0.319 0.521 0.561 Gastric- 6.09 ± 6.09 ± 6.10 ±6.17 ± 6.15 ± nemius 0.37 0.37 0.45 0.32 0.41 muscle index P value 0.9950.973 0.428 0.582 Serum 56.76 ± 58.59 ± n/a n/a 44.95 ± glycerinate24.33 19.15 15.76 (mg/dl) P value 0.756 0.040 Serum free 151.08 ± 163.36± n/a n/a 209.45 ± fatty acid 79.12 82.92 125.76 (umol/l) P value 0.5730.041 Serum 1.89 ± 1.73 ± n/a n/a 1.68 ± leptin 0.66 0.63 0.52 (ng/ml) Pvalue 0.29 0.18 Serum 25.78 ± 20.11 ± n/a n/a 22.49 ± insulin 7.63 4.875.83 (uU/ml) P value 0.0017 0.0882C. Discussion and Conclusion

The above results indicate that the rats fed with a diet added with 75ppm antibodies had 16.89% reduction in parametrial fat, 7.76% reductionin mesenteric fat, 11.88% reduction in perirenal fat and 12.60 greduction celiac fat compared with the control rats. The rats fed withthe diet added with 75 ppm antibodies had no noticeable difference ingastrocnemius muscle weight compared with the control rats.

The above results indicate that the rats fed with a diet added with 6000ppm antibodies had 14.90% reduction in parametrial fat, 10.56% reductionin mesenteric fat, 20.21% reduction in perirenal fat and 13.05 greduction celiac fat compared with the control rats. The rats fed withthe diet added with 6000 ppm antibodies had 1.08% increase ingastrocnemius muscle weight compared with the control rats. The increasewas however insignificant.

The experimental data suggests that the amount of food intake by thedifferent groups of rats was about the same statistically.

The experimental data suggest that the antibodies prepared andadministered in accordance with the present invention is effective inmodulating and in particular reducing the adipose tissues in a targetanimal.

Based on the findings of the above experiments, the antibodies whenadministered in animal farming (e.g. via an animal feed) can produceanimals with leaner meat. Among most farm animals for producing meat forhuman consumption, swine tend to have a rather high fat content. Thus,the present invention is particularly suitable to be applied in raisingswine.

When applied for use in humans, the antibodies can be used in themanufacture of a medicament or composition for the treatment orprevention of obesity and/or related conditions. Alternatively, theantibodies can be added to a food supplement suitable for consumption byhumans. A medicament comprising such antibodies may also be produced.

The contents of each of the references mentioned above and UK patentapplication no. 0212876.7 are herein incorporated by reference in theirentirety. It is to be noted that numerous variations, modifications, andfurther embodiments are possible and accordingly, all such variations,modifications and embodiments are to be regarded as being within thescope of the present invention and to be understood by the personsskilled in the art.

1. A non-therapeutic method of producing antibodies that bind adiposetissues in a target animal in need of said antibodies for modulating thecontent of adipose tissues in said target animal, the method comprisingthe steps of: (i) preparing an antigen from adipose tissues of a sourceanimal wherein said source animal and said target animal belong toclosely related species; (ii) administering said antigen to an egglaying animal to cause production of said antibodies wherein saidegg-laying animal and said target animal belong to different species;and (iii) obtaining said antibodies from eggs of said egg-laying animalwherein said egg-laying animal and said source animal belong todifferent species.
 2. A method according to claim 1 comprising a step ofcausing production of said antibodies from within the body of saidegg-laying animal.
 3. A method according to claim 1 or 2 comprising astep of causing deposition of said antibodies to said eggs of saidegg-laying animal.
 4. A method according to any preceding claim whereinsaid step of obtaining comprises a step of isolating said antibodiesfrom the egg yolk of said eggs.
 5. A method according to any precedingclaim wherein said antigen comprises plasma membrane, its adipocyteplasma membrane surface proteins, or fragments thereof, of said adiposetissues of said source animal.
 6. A method according to any precedingclaim wherein said antibodies are polyclonal antibodies.
 7. A methodaccording to any preceding claim wherein said source animal and saidegg-laying animal belong to distinctly different species.
 8. A methodaccording to any preceding claim wherein said target animal and saidegg-laying animal belong to distinctly different species.
 9. A methodaccording to any preceding claim wherein said target animal is a farmanimal.
 10. A method according to any preceding claim wherein saidsource animal and/or said target is a swine.
 11. A method according toany one of claims 1 to 8 wherein said target animal is a patient.
 12. Amethod according to any preceding claim wherein said egg-laying animalin an avian animal and said source animal is a non-avian animal.
 13. Amethod according to any preceding claim wherein said source animal is amammal.
 14. A method according to any preceding claim wherein one ormore of said animals is sacrificed after said method.
 15. Antibodiesobtainable according to the method defined in any one of claims 1 to 14.16. Antibodies according to claim 15 for use in a method of treatment ordiagnosis.
 17. Use of antibodies defined in claim 15 or 16 for themanufacture of a medicament for the treatment of a condition caused byan excess of adipocytes.
 18. A feed additive comprising an effectiveamount of antibodies defined in claim 15 or
 16. 19. A feed additiveaccording to claim 18 adapted to lower the content of said adiposetissues in said target animal.
 20. A feed additive according to claim 18or 19 wherein said composition comprises egg yolk of said eggscontaining said antibodies.
 21. A medicament comprising apharmaceutically effective amount of antibodies defined in claim 15 or16.
 22. A medicament according to claim 21 adapted to be administeredvia ingestion.
 23. A medicament according to claim 21 adapted to beadministered via injection.
 24. A method of modulating content ofadipose tissues in the body of a target animal in need of antibodies,comprising the steps of: (i) preparing an antigen from adipose tissuesof a source animal; (ii) administering said antigen to an egg-layinganimal; (iii) allowing said antibodies to be produced by said egg-layinganimal in response to said antigen; (iv) obtaining said antibodies fromeggs of said egg-laying animal; and (v) administering a pharmaceuticallyeffective amount of said antibodies to said target animal by ingestion,wherein said source animal and said egg-laying animal belong todifferent species.
 25. A method according to claim 24 wherein said stepof obtaining comprises a step of isolating said antibodies from the eggyolk of said eggs.
 26. A method according to claim 24 or 25 comprising astep of binding said antibodies to characterizing components of plasmamembrane of said adipose tissues in said target animal.
 27. A methodaccording to claim 24, 25 or 26 comprising a step of binding saidantibodies to granular viscosity proteins of said adipose tissues insaid target animal.
 28. A method according to claim 24, 25 or 26comprising a step of binding said antibodies to fiber viscosity proteinsof said adipose tissues in said target animal.
 29. A method according toany one of claims 24 to 28 wherein said antigen comprises plasmamembrane, its adipocyte plasma membrane surface proteins, or fragmentsthereof, of said adipose tissues of said source animal.
 30. A methodaccording to any one of claims 24 to 29 wherein said antibodies arepolyclonal antibodies.
 31. A method according to any one or claims 24 to30 wherein said source animal and said egg-laying animal belong todistinctly different species.
 32. A method according to any one ofclaims 24 to 31 wherein said target animal and said egg-laying animalbelong to different species.
 33. A method according to claim 32 whereinsaid target animal and said egg-laying animal belong to distinctlydifferent species.
 34. A method according to any one claims 24 to 33wherein said target animal and said source animal belong to a samespecies.
 35. A method according to any one claims 24 to 33 wherein saidtarget animal and said source animal belong to a closely relatedspecies.
 36. A method according any one of claims 24 to 36 wherein saidmethod is a non-therapeutic method.
 37. A method according to any one ofclaims 24 to 36 wherein said target animal is a farm animal
 38. A methodaccording to any one of claims 24 to 37 wherein said source animaland/or said target is a swine.
 39. A method according to any one ofclaims 24 to 36 wherein said target animal is a patient.
 40. A methodaccording to any one of claims 24 to 39 wherein said egg-laying animalin an avian animal and said source animal is a non-avian animal.
 41. Amethod according to any one of claims 24 to 40 wherein said sourceanimal is a mammal.
 42. A method according to any one of claims 24 to 41wherein one or more of said animals is sacrificed after said method. 43.A method of producing antibodies that bind adipose tissues in a targetanimal in need of said antibodies for modulating the content of adiposetissues in said target animal, the method comprising the steps of: (i)preparing an antigen from adipose tissues of a source animal whereinsaid source animal and said target animal belong to closely relatedspecies; (ii) administering said antigen to an egg-laying animal tocause production of said antibodies wherein said egg-laying animal andsaid target animal belong to different species; and (iii) obtaining saidantibodies from eggs of said egg-laying animal wherein said egg-layinganimal and said source animal belong to different species.